-
The invention relates to the targeted delivery of
substances to cells.
-
Delivery of substances to cells allows specific
treatment of said cells with compounds that act in the
targeted cell. For example, tumour cells, when targeted with
toxic components, selectively die when said toxin is
delivered to said cell. Yet other cells, when provided with a
gene lacking in said cell, can be restored in their function,
so-called gene therapy.
-
Delivery of a compound to a cell preferably occurs with
a vehicle or particle that effectively brings the compound to
the desired cell or cells and than delivers said compound
into that cell (in vivo or in vitro) where it can exert its
action. For this purpose, for example particles such as
virus-like particles are suited. These particles, often
derived from known viruses, such as retrovirus or adenovirus,
are small enough to penetrate in-between tissues and cells
and arrive at a cell of choice where it for example can fuse
with said cell and deliver its compound. Said virus-like
particles may or may not be infectious in themselves, their
main concern is the targeted delivery of the compound of
interest, such as a gene, a toxin or immunostimulating
components such as antigens. Yet other examples are gene-delivery
vehicles, specifically designed to transfer a gene
to a cell of interest. Virus-like particles capable of
delivering a gene are examples of said gene-delivery
vehicles, however, also other examples of such vehicles, of
non-viral origin, such as liposomes or microbodies, or even
latex particles, are known. Vehicles such as liposomes or
microbodies can of course also carry other compounds than a
gene, in-particular toxic or immunostimulating components
such as antigens can be included in such a vehicle.
-
These vehicles or particles all have in common that they
need to be provided with a molecule or fragment thereof
(ligand) capable of binding with said targeted cell, allowing
targeting of said particle or vehicles to cells. There is a
need for specific or broadly applicable ligands that react
with cell-surface receptors on cells. In particular there is
a need for ligands that react with cell-surface receptors
after which efficient transfer of said compound to said cell,
such as a gene, is possible. Especially in human medicine,
such a ligand would enable better application of gene-transfer
therapy than is possible now.
-
It has been a long-standing objective to exploit
retrovirus technology in human gene therapy applications.
However, the infection spectrum of retroviruses clearly
limits the applications of these viruses in such
applications. All known env variants have a rather broad
infection spectrum in common. Here lies one of the major
shortcomings of current recombinant retrovirus technology.
For the purpose of gene therapy, retroviruses are very useful
vehicles for the transfer of therapeutic sequences, if proper
ligand-receptor targets were available. The concept of the
use of retroviruses or adenoviruses in human gene therapy is
well documented. However, it would be clearly advantageous
and desirable to devise a strategy for targeted delivery of
virus-like particles or gene delivery vehicles and
modification of the infection spectrum.
-
The invention provides a method for selecting at least
one mutant protein derived from a viral protein as a ligand
capable of binding to a cell-surface receptor, comprising
displaying at least one mutant of said protein on the surface
of a micro-organism expressing said protein, further
comprising selecting at least one such a mutant microorganism
for its capacity to bind to said receptor. For
example provided are materials and methods to develop
envelope or capsid molecules that can bind to preferred
target cells for gene therapy. In order to obtain a novel
protein capable of binding a chosen target, DNA molecules,
each encoding for example a envelope molecule or parts
thereof comprising one of a family of similar potential
binding domains and a structural signal calling for the
display of the protein on the outer surface of a micro-organism,
such as a bacterial cell, bacterial spore or phage
(the genetic package) are introduced into that genetic
package. The protein or part thereof is expressed and the
potential binding domains displayed on the outer surface of
the package. The micro-organisms, cells or viruses bearing
the binding domains which recognise the target receptor
molecule are isolated and amplified. The successful binding
domains of the variant proteins are then characterised. One
or more of these successful binding domains are used as a
basis for the design of a new family of potential binding
domains, and the process is repeated until a novel binding
domain having a desired affinity and specificity for the
target molecule is obtained.
-
The invention provides a method according to the
invention further comprising incorporating said protein in a
virus-like particle or gene-delivery vehicle allowing
targeting of said virus-like particle or gene-delivery
vehicle to cells carrying said receptor. Mutant proteins
find applications in the design of vector systems for entry
into cells including human cells. Preferred are those
retroviral envelope molecules or virus capsid proteins or
parts thereof, which - when incorporated in a virus-like
particle or gene delivery vehicle - can infect desired target
cells at increased specificity and efficiency.
-
The invention further provides a method according to the
invention wherein said micro-organism is a filamentous phage.
Phage display is a powerful technique allowing the efficient
screening of large numbers of random sequences in order to
select the best binding sequence for any chosen application.
The power of phage display is based on the ability of
filamentous bacteriophages to easily express and display
foreign protein moieties as fusions with phage coat proteins
accompanied by the genetic material encoding that foreign
protein moiety. Foreign sequences can be inserted at the
amino terminus of either gene III (encoding for the minor
coat protein) or gene VIII (encoding for the major coat
protein) of a filamentous phage. This is usually possible
without significantly interfering with the phage's life cycle
(Smith, 1985),US patents 5,223,409 and 5,403,484. In one
embodiment of the invention the genetic package is a
filamentous phage such as a M13 phage, and the protein
includes for example the outer surface transport signal of
the M13 gene III protein.
-
The invention further provides a method according to the
invention wherein said protein comprises at least a fragment
of an envelope protein, preferably derived from a retrovirus.
The invention provides retroviral vectors including murine
leukemia retroviral and lentiviral vectors. Peptide phage
display has been used for the isolation of ligands that can
be incorporated in a retroviral envelope to change the
tropism of the original envelope and the resulting retroviral
vector (Hall et al., 1997). The disadvantage of this approach
is that the effect of insertion of a foreign peptide into a
retroviral envelope is unpredictable and therefore the
general applicability for retroviral vector improvement
limited. Therefore functional display of the retroviral
envelope itself is highly preferred.
-
The invention provides functional display of a
retroviral envelope mutant on the surface of a filamentous
phage. An advantage is that said mutant is presented in its
most natural conformation, allowing for unrestrained
selection of mutants capable of binding to said receptor. In
one embodiment of the invention a potential binding domain or
ligand is related to ecotropic Moloney Murine Leukemia
envelope protein, the genetic package is M13 phage, and the
protein includes the outer surface transport signal of the
M13 gene III protein.
-
In yet another embodiment, the invention provides a
method according to the invention wherein said protein
comprises at least a fragment of a capsid protein, preferably
derived from an adenovirus. Said capsid protein can for
example comprise a selected ligand as provided by the
invention and be derived from adenovirus icapsid protein
including but not limited to the HI loop of the knob domain
of an adenovirus, preferably an adenovirus which does not
bind to the adenoviral receptor CAR1 or MHC1. This results in
an adenovirus that enters cells through a receptor of choice,
thereby greatly facilitating the usefulness of a virus-like
particle or gene-delivery vehicle derived from adenovirus.
-
In yet another embodiment of the invention, a method
according to the invention is provided wherein said protein
comprises a at least a fragment of a coat protein, preferably
derived from a bacteriophage.
-
The invention provides a method according to the
invention wherein said cell-surface receptor is an amino acid
transporter, preferably a cationic amino acid transporter
such as mCAT1. Selection for protein mutants according to the
invention with preferred characteristics from phage libraries
can be done using in vitro or in vivo selection methods or a
combination of both. Phage libraries displaying mutant
envelopes can be subjected to a number of selection rounds on
desired target cells or tissues by injecting the libraries
intravenously animals such as mice in vivo combined with a
selection on relevant human tissues. By way of illustration
and not intended to be a limitation human chimeric NOD-SCID
mice (Dick et al., 1997) which have a functional human
hemopoiesis can be used to inject envelope display libraries
followed by sampling the bone marrow and isolation of the
targeted phages from marrow cells after various periods of
time. This is then followed by amplification of the envelope
displaying phages in E.coli and repeated in vivo selections.
The in vivo selections can be combined with selections on
purified samples of human hemopoietic stem cells including
but not limited to CD34positive CD38 negative or
CD34positive(CD33,CD38,CD71)negative cells isolated from human
bone marrow, cord blood or growth factor mobilised peripheral
blood using flow cytometry (Knaan-Shanzer et al., 1996).
-
The invention further provides a method wherein said
mutant protein is derived mutant retroviral envelopes that
are derived from wild-type envelopes including but not
limited to ecotropic, amphotropic and GALV retroviral
envelopes by employing the disclosed envelope display
technology and which employ a novel or better receptor
molecule to enter the target cell by binding or improved
binding to this novel or native receptor or receptors. These
new retroviral envelope molecules, when incorporated in a
virus-like particle or gene-delivery vehicle, can bind to
receptor positive cells, such as human PHSCs, with increased
specificity and efficiency. Using methods described herein
other retroviral envelope molecules can be displayed and
converted into libraries of mutant envelopes including but
not limited to GALV, amphotropic envelope and spleen necrosis
viral envelope (Battini et al., 1992),(Dornburg, 1995). The
mutant retroviral envelopes can be used to pseudotype
recombinant type C retrovirus including but not limited to
murine leukemia retroviral vectors. In a preferred example,
said mutant protein is derived from glycoprotein 70 (gp70) of
Moloney murine leukemia virus; constructs comprising such
mutant protein are for example provided in the experimental
part of this description.
-
The invention further provides a virus-like particle or
gene-delivery vehicle obtainable by a method according to the
invention. Examples of such particles or vehicles are derived
from retrovirus, adenovirus or bacteriophage M13, as
described above. Said particles or vehicles can further
comprise toxins or antigens or other substances that need to
be delivered to a cell, be it in vitro or in vivo.
-
In a preferred embodiment, a virus-like particle or
gene-delivery vehicle is provided for use as a gene-transfer
vehicle. Another embodiment of this invention is to use the
disclosed retroviral envelope display technology to develop
particles or vehicles such as retroviral envelope mutants
derived from pathogenic retroviruses and which can be used to
block productive infection of cells in a human patient.
Mutant envelope displaying phages that block a receptor can
then be used as such or recombinant soluble mutant protein
can be made thereof and used to treat pathogenic virus
infections.
-
Another embodiment of this invention is to employ the
envelope mutants made according tot the methods described
herein in non-viral gene transfer vehicles including but not
limited to incorporation in liposomes and phosphazenes
(W097/07226). This can be done by producing mutant envelopes
as recombinant proteins or as envelope displaying phages and
mixing or chemically linking them with liposomal or
phosphazene preparations carrying a DNA expression construct
of a therapeutic DNA sequence. Alternatively the
retroviral envelope displaying phages itself may be used as
the vector for the therapeutic sequence and modified to a DNA
vector.
-
The invention further provides a method for selecting a
filamentous phage expressing a protein capable of binding to
a ligand comprising constructing a phage library, enriching
said library for phages having desired binding
characteristics by at least one round of selection of phages
for their capacity to bind to a synthetic peptide derived
from said ligand, further comprising enriching said library
for phages having desired binding characteristics by at least
one round of selection of phages for their capacity to bind
to a cell expressing said ligand, for example wherein said
ligand is a amino acid transporter, preferably mCAT1.
-
The invention is further explained in the experimental
part not limiting the invention thereto.
Experimental part
-
Retroviruses are RNA viruses which efficiently integrate
their genetic information into the genomic DNA of infected
cells via a reverse-transcribed DNA intermediate. This
property of their life-cycle and the fact that parts of their
genetic material can be replaced by foreign DNA sequences
make retroviruses one of the most promising vectors for the
delivery of genes in human gene therapy procedures, most
notably for gene therapies which rely on gene transfer into
tissues where integration of the therapeutic sequence is
needed. Most retroviral vector systems are based on mouse
retroviruses and consist of two components, i.e. (i) the
recombinant retroviral vector carrying the foreign sequences
of interest, and (ii) so-called packaging cells expressing
the structural viral proteins of which the encoding sequences
are lacking in the retroviral vector. Expression of (i) in
(ii) results in the production of recombinant retroviral
particles capable of transducing susceptible target cells.
-
The infectivity and host cell range (tropism) of the
retrovirus particle is conferred by an envelope glycoprotein
expressed in the bilayer of the retroviral particle and which
specifically binds to a receptor molecule on the target cell
membrane. The envelope glycoprotein of all known retroviruses
consists of two associated peptides gp70 and p15 and which
are derived by proteolytic cleavage from the same precursor
protein encoded by the retroviral envelope (env) gene
(Gunzburg and Salmons, 1996; Weiss, 1996). The amino terminal
domain gp70 encompasses specific binding site(s) for its
receptor on the target cell membrane, determining the virus
host range. Within this domain of about 200 amino acids
highly conserved sequences are present that are interrupted
by two segments designated VRA and VRB which vary in sequence
and length among various mammalian type C retroviruses. In
addition some retroviral envelopes have an additional
variable domain VRC (Battini et al., 1992). The carboxy
terminal peptide or pl5 polypeptide is assumed to mediate
fusion between the virus membrane and - depending on the type
of virus - the plasma membrane or intracellular vesicle
membrane of the target cell (Januszeski et al., 1997; Thomas
et al., 1997).
-
Recently also N-terminal sequences have been implied to
play a role in membrane fusion in addition to the key
determinant of membrane fusion, the pl5 polypeptide (Bae et
al., 1997). Besides its role in fusion between the virus
membrane and target cell membranes, the carboxy terminal
peptide contains trans-membrane anchor sequences for
anchoring in the virion bilayer, and is also crucial for the
selective uptake of the envelope glycoprotein in the virus
particle. The envelope oligomeric complex consisting of gp70
and p15 is expressed on the surface of the virion as a
trimer. Trimerization is believed to be crucial for membrane
fusion and is mediated by a trimerization domain in p15 on
the outside of the virion (Zhao et al., 1997),(Fass et al.,
1996). Nevertheless receptor binding has been observed for
monomeric soluble forms containing the variable regions of
ecotropic and amphotropic gp70 proteins (Davey et al., 1997).
It has been a long-standing objective to exploit retrovirus
technology in human gene therapy in order to transfer
therapeutic sequences. However, the infection spectrum of
retroviruses limits the applications of these viruses in such
applications. In numerous gene therapy applications improved
or targeted delivery of genes into defined cells or tissues
would be desired, including in vitro gene transfer into cell
types that are present in low abundance in cell mixtures and
in vivo gene transfer into specific cells and tissues of
patients and animals.
-
Several envelope glycoprotein variants with different
infection spectra for mammalian cells have been identified
(Battini et al., 1992).
Many envelope variants have a rather broad infection spectrum
in common or have infection spectra that do not include human
or primate cells and thus are not relevant for human gene
therapy applications. Those envelope molecules that do infect
primate cells when incorporated in a recombinant retroviral
vector often transduce their specific target cells with low
efficiencies. Here lies one of the major shortcomings of
current recombinant retrovirus technology.
-
Examples of recombinant viruses that infect primate cells are
virions that carry amphotropic or GaLV envelopes. Recombinant
viruses carrying an amphotropic or GaLV envelope are capable
of infecting human and murine cells and are commonly used in
gene transfer trials including human gene therapy involving
the pluripotent hemopoietic stem cell (PHSC) an important
target for human gene therapies (Havenga et al., 1997). Gene
transfer frequencies into PHSCs of human patients and non
human primate animal models have been shown to be extremely
low and limit therapeutic stem cell gene therapy (Havenga et
al., 1997; Hoogerbrugge et al., 1996; Van Beusechem et al.,
1993; van Beusechem et al., 1992).
-
One limiting factor has indeed been shown to be low
expression levels of retroviral receptors such as the
receptor mediating entry of amphotropic MuLV retrovirus
(GLVR2) (Orlic et al., 1996; van Es et al., 1996). The
quiescent state of PHSCs when isolated for ex vivo gene
transfer procedures poses another blockade (Knaan-Shanzer et
al., 1996). Murine stem cell gene therapy experiments have
traditionally been performed with ecotropic MuLV vectors
(Havenga et al., 1997). Recombinant viruses carrying an
ecotropic envelope are only capable of infecting murine
cells. Transfer of genes into murine PHSCs using ecotropic
retroviral vectors has been shown to result in high
transduction efficiencies in circulating PHSC derived
peripheral blood cells (PBL). The transduction efficiencies
are high enough to be therapeutic if achieved in human PHSCs,
reaching levels of PHSC gene transfer varying between 30-80
%.
-
A small number of studies have been performed in which the
transduction efficiency into murine PHSCs of ecotropic and
amphotropic retroviruses were actually compared directly
(Havenga et al., 1997). One of these studies indicated that
infection with amphotropic retrovirus resulted in expression
and thus transgene presence for less than 8 weeks whereas
infection with ecotropic virus resulted in expression for
more than 44 weeks after transplantation (Demarquoy, 1993).
In a similar study, ecotropic virus was shown to be
approximately 50 fold more efficient in transducing murine
PHSCs as compared to amphotropic retrovirus (Orlic et al.,
1996).
-
Ecotropic and amphotropic retrovirus differ in the receptor
that is employed for virus entry (Albritton et al., 1989; van
Zeijl et al., 1994). Ecotropic virus binds target cells via
the ecotropic receptor mCAT1 which is a transporter of
cationic L-amino acids (Kim et al., 1991) and amphotropic
retrovirus binds target cells via the amphotropic receptor
GLVR2, a sodium dependent phosphate transporter (Kavanaugh et
al., 1994; Miller and Miller, 1994; van Zeijl et al., 1994).
-
Another limitation for the application of retroviral vectors
in gene therapy for human diseases is their relative
instability during production, down stream processing
including purification and storage of final retroviral gene
therapeutics or vaccins (Braas et al., 1996). The in vitro
half life of recombinant murine leukemia retroviral vectors
is only in the range of 8 hours (Kaptein et al., 1997).
In vivo administration of retroviral vectors in particular
those based on murine leukemia retrovirus is hampered by
inactivation by the immune-system including the complement
system and would greatly benefit from technologies enabling
rapid improvement of envelopes as envelopes are a prime
target for the immune-system (Takeuchi et al., 1994).
Retroviral envelopes can be given a defined specificity for a
specific ligand by insertion or inclusion of that ligand.
Ligands that have been used are synthetic peptides (Hall et
al., 1997), growth factors (Cosset et al., 1995), single
chain antibodies (Ager et al., 1996; Marin et al., 1996) and
immunoglobulin binding ligands such as one of the receptors
for immunoglobulins (W097/05266). To produce envelopes that
when expressed in the retroviral bilayer membrane give rise
to functional, high titer vector preparations is difficult
and cumbersome because the effect of insertion of a foreign
sequence into the envelope is unpredictable. The envelope of
spleen necrosis virus is an envelope that in general allows
easy insertion of single chain antibodies but the resulting
specificity of the molecule is in doubt (Chu et al., 1994).
-
A number of mutant ecotropic envelope molecules have been
described in the literature. MacKrell et al have mutated
amino acids within the receptor-binding domain VRA of
ecotropic MuLV envelope in order to identify residues
involved in receptor binding. Virions incorporating mutant
envelopes carrying mutations at amino acid residue D84 have
lost their binding capabilities to the ecotropic receptor
mCAT1 (MacKrell et al., 1996). Virions carrying D84 mutated
envelope protein were tested on human cells to search for a
possible change in receptor recognition specificity but were
found not to infect human cells (Mike Januszeski, personal
communication).
Skov and Andersen have studied ecotropic Moloney envelope
interactions with mCAT1 by generation of mutant envelope
molecules with mutated arginine and lysine residues in gp70
including VRA followed by introduction in a replication
competent retroviral backbone (Skov and Andersen, 1993).
Mutations R135G, K137Q, R157G and R159A (R102G,K104Q,R124G
and R126A without signal peptide respectively) resulted in
virions that were not able to replicate.
Kingsman et al have described in PCT application WO96/31602
an insertion site in the VRA domain of ecotropic envelope
which allows modification of the tropism. An integrin binding
sequence was inserted resulting in infection of human cells
expressing the respective integrin other peptides inserted at
this site did not result in useful molecules.
PVC-211 murine leukemia virus (MuLV) is a neuropathogenic
variant of ecotropic Friend MuLV (F-MuLV) that causes a
rapidly progressive neurodegenerative disease in susceptible
rodents. PVC-211 MuLV, but not the parental F-MuLV, can
infect rat brain capillary endothelial cells (BCEC)
efficiently, and the major determinant for BCEC tropism of
PVC-211 MuLV is localised within the envelope gene. More
specific analysis indicated that E116G and E129K
substitutions in the background of the F-MuLV envelope
envelope protein were sufficient for conferring BCEC tropism
on the virus (Masuda et al., 1996a). Host range changes of
these mutations were found to include CHO cells normally not
infectable with ecotropic F-MuLV or M-MuLV. The latter
suggests that these mutations overcome a negative effect of
CAT1 CHO cell receptor glycosylation in the region of virus
binding in the third extracellular domain of mCAT1 (Masuda et
al., 1996b).
In conclusion murine leukemia retroviral envelope mutants
revealed sites in the envelope that are important for folding
or more importantly for receptor binding but failed to yield
variants useful for human gene therapy purposes.
-
By employing particular natural retroviral envelope variants
the transduction spectrum can be limited or expanded to some
extend, but true specificity for particular human target
cells of interest can not be obtained following the above
described strategy (Masuda et al., 1996a; von Kalle et al.,
1994; Wilson et al., 1994).
-
Evolution of retroviruses to variants with preferred tropism
characteristics without the use of rational approaches
including specific ligand insertions or mutagenesis of
specific sites can be done using replication competent
retroviruses which are evolved for particular target cells by
multiple passages of the virus on those target cells. The
disadvantages of this type of approach are: (1) The process
is lengthy in time and laborious, (2) dependant on
replication and not only on the envelope proteins that need
to be evolved or optimized and (3) often the virus needs to
have a low but clear affinity for the target cells in
question (Taplitz and Coffin, 1997), (Yoshikura, 1975),
(Jolicoeur, 1978; Jolicoeur, 1979), W097/22709.
-
Phage display is a powerful technique allowing the efficient
screening of large numbers of random sequences in order to
select the best binding sequence for any chosen application.
The power of phage display is based on the ability of
filamentous bacteriophages to easily express and display
foreign protein moieties as fusions with phage coat proteins
accompanied by the genetic material encoding that foreign
protein moiety. Foreign sequences can be inserted at the
amino terminus of either gene III (encoding for the minor
coat protein) or gene VIII (encoding for the major coat
protein) of a filamentous phage. This is usually possible
without significantly interfering with the phage's life cycle
(Smith, 1985),US patents 5,223,409 and 5,403,484. Phage
display has been used successfully for the display of
libraries of mutant immunoglobulin derived protein domains
including single chain antibodies (Clackson et al., 1991; de
Kruif and Logtenberg, 1996), (Stausbol-Gron et al., 1996).
Phage display libraries displaying random peptides have been
used extensively for the selection of epitope mapping (Jellis
et al., 1993), tissue specific peptides (Heiskanen et al.,
1997) and in vivo isolation of tissue specific peptides that
bind and internalise (Pasqualini and Ruoslahti, 1996). Other
molecules that have been displayed as libraries of randomised
sequences are for example the HIV receptor CD4 (Roberts et
al., 1996), interleukin 2 (Cabibbo et al., 1995) and
plasminogen activator (Pannekoek et al., 1993). Display of
these important molecules enabled the isolation of mutants
with preferred and novel characteristics for application in
medicine.
-
As described above easy and rapid procedures to isolate
envelope variants would greatly facilitate the development of
improved retroviral vectors including murine leukemia
retroviral and lentiviral vectors. Peptide phage display has
been used for the isolation of ligands that can be
incorporated in a retroviral envelope to change the tropism
of the original envelope and the resulting retroviral vector
(Hall et al., 1997). The disadvantage of this approach is
that the effect of insertion of a foreign peptide into a
retroviral envelope is unpredictable and therefore the
general applicability for retroviral vector improvement
limited. Therefore functional display of the retroviral
envelope itself is highly preferred. We disclose the
functional display of a retroviral envelope on the surface of
a filamentous phage. The subject of this invention opens up a
number of possibilities for the development of improved
retroviral, non-viral and phage vectors including lentiviral
vectors by making libraries of displayed retroviral envelope
mutants and selecting appropriate mutant envelopes by in
vitro, in situ or in vivo screening.
-
In conclusion, the concept of the use of retroviruses in
human gene therapy is well documented (Gordon and Anderson,
1994; Havenga et al., 1997; Vile et al., 1996). However, it
would be clearly advantageous and desirable to devise a
strategy for targeted delivery of retroviruses, and
modification of the infection spectrum by using phage display
of libraries of functional retroviral envelopes.
In the present invention we describe the methodology and
materials to rapidly and efficiently generate mutant
retroviral envelope molecules including but not limited to
ecotropic murine leukemia retrovirus envelope proteins. These
methods and materials are extremely useful for the fields of
gene therapy and virology.
-
The present invention discloses examples of methods to
develop novel retroviral envelope sequences that are
optimised for desired target cells including but not limited
to hemopoietic stem cells. We have developed retroviral
envelope display using a filamentous phage. We have used the
ecotropic Moloney murine leukemia retroviral envelope
sequence as a model retroviral envelope molecule. Five
different constructs were made encompassing various parts of
the gp70 receptor binding moiety of the envelope. In all 5
constructs the retroviral envelope signal peptide was
replaced by the pelB leader sequence of m13 gpIII. All
constructs displayed gpIII/envelope fusion protein as
addressed by Western blotting of envelope phages. Those
constructs encompassing variable regions A and B plus or
minus the polyproline hinge region were found to efficiently
bind to mCAT1 expressing human 911 cells and not to control
cells and thus displayed functional gp70. The construct
displaying all of gp70 did exhibit functionality but to a
lesser extent. Finally, constructs that encode only variable
region A and B or only A did not show binding to mCAT1 cells.
Infection of mCAT1 expressing human cells with ecotropic
retrovirus decreased the binding of the envelope displaying
phages confirming specific binding of the envelope displaying
phages to the ecotropic retroviral receptor. This invention
opens up a number of possibilities for the development of
improved retroviral vectors including lentiviral vectors by
making libraries of displayed envelope mutants and selecting
appropriate mutant envelopes by in vitro, in situ or in vivo
screening.
-
The methodology uses filamentous phage display of retroviral
envelope molecules or parts thereof that can be used to
develop gene transfer vehicles such as retroviral,
lentiviral, phage gene transfer or non-viral gene transfer
vehicles.
-
Included in the present invention are filamentous phages
displaying mutant envelope molecules that can be used to
transfer genes into cells by modification of the phage genome
using techniques known in the art. This would combine the
ease of producing bacteriophages with the highly efficient
binding and entry of mammalian cells by mutant retrovirus
envelopes.
-
Selection for protein mutants with preferred characteristics
from envelope libraries disclosed in this application can be
done using in vitro or in vivo selection methods or a
combination of both. Phage libraries displaying mutant
envelopes can be subjected to a number of selection rounds on
desired target cells or tissues by injecting the libraries
intravenously into the bloodstream of animals including but
not limited to mice in vivo combined with a selection on
relevant human tissues.
By way of illustration and not intended to be a limitation
human chimeric NOD-SCID mice (Dick et al., 1997) which have a
functional human hemopoiesis can be used to inject envelope
display libraries followed by sampling the bone marrow and
isolation of the targeted phages after various periods of
time. This is then followed by amplification of the envelope
displaying phages in E.coli and repeated in vivo selections.
The in vivo selections can be combined with selections on
purified samples of human hemopoietic stem cells including
but not limited to CD34positive CD38 negative or
CD34positive (CD33, CD38, CD71)negative cells isolated from human
bone marrow, cord blood or growth factor mobilised peripheral
blood using flow cytometry (Knaan-Shanzer et al., 1996).
-
Preferred are mutant retroviral envelopes that are derived
from wild-type envelopes including but not limited to
ecotropic, amphotropic and GALV retroviral envelopes by
employing the disclosed envelope display technology and which
employ a novel or better receptor molecule to enter the
target cell by binding or improved binding to this novel or
native receptor or receptors. These new retroviral envelope
molecules, when incorporated in a retroviral virion, will be
able to infect the receptor positive cells such as human
PHSCs with increased specificity and efficiency. Using the
methods described herein other retroviral envelope molecules
can be displayed and converted into libraries of mutant
envelopes including but not limited to GALV, amphotropic
envelope and spleen necrosis viral envelope (Battini et al.,
1992),(Dornburg, 1995). The mutant retroviral envelopes can
be used to pseudotype recombinant type C retrovirus including
but not limited to murine leukemia retroviral vectors.
In a further embodiment of the present invention these new
mutant envelopes can be used to pseudotype lentiviral vectors
including equine or HIV derived lentiviral vectors (Kafri et
al., 1997; Kim et al., 1998; Poeschla et al., 1996; Rizvi and
Panganiban, 1992),(Miyoshi et al., 1997; Naldini et al.,
1996b).
-
In a further embodiment of the present invention envelopes
with ligands inserted such as a growth factor or a synthetic
peptide and which function in a retrovirion but insufficient
to be useful can be displayed using the technology disclosed
in this invention. Display of libraries of randomised or
mutagenized versions of these ligand containing envelopes
will enable the selection of improved variants while
retaining the ligand mediated specificity. An example of such
a envelope is disclosed in W097/05266 and is an ecotropic
envelope containing an immunoglobulin binding domain of a Fc
receptor.
-
In a further embodiment of this invention retroviral envelope
display can be used to evolve retroviral envelopes in vivo
and in vitro to envelopes that are less sensitive i.e.
resistant to the host's immune system. This application
enables further improvement and applicability of retroviral
vectors.
-
Any mutant envelope molecules or parts thereof made according
to the methods described herein can then be ligated into full
length mammalian retroviral envelope expression constructs
and introduced in cell lines expressing and containing all
the sequences necessary for the generation of infectious and
functional retroviral particles including but not limited to
cell lines that express murine leukemia gag-pol constructs
and a retroviral vector containing long terminal repeats
(LTRs),and retroviral RNA packaging signals such as those
vectors described in WO96/35798. The mutant envelopes made
according to the subject material of this invention can also
be used to pseudotype vectors other than murine leukemia
retroviral vectors including but not limited to lentiviral
vectors (Naldini et al., 1996a).
Another embodiment of this invention is to use the disclosed
retroviral envelope display technology to develop retroviral
envelope mutants derived from pathogenic retroviruses and
which can be used to block productive infection of cells in a
human patient. Mutant envelope displaying phages that block a
receptor can then be used as such or recombinant soluble
mutant protein can be made thereof and used to treat
pathogenic virus infections.
-
Another embodiment of this invention is to employ the
envelope mutants made according tot the methods described
herein in non-viral gene transfer vehicles inlcuding but not
limited to incorporation in liposomes and phosphazenes
(WO97/07226). This can be done by producing mutant envelopes
as recombinant proteins or as envelope displaying phages and
mixing or chemically linking them with liposomal or
phosphazene preparations carrying a DNA expression construct
of a therapeutic DNA sequence. Alternatively the retroviral
envelope displaying phages itself may be used as the vector
for the therapeutic sequence and modified to a DNA vector.
-
The skilled artisan will be able to apply the teaching of the
present invention to other virus capsid or envelope or non-viral
gene transfer molecules or vehicles than those
exemplified herein without departing from the present
invention and therefore the examples presented are
illustrations and not limitations. It is intended that all
such other examples be included within the scope of the
appended claims.
Example 1. Construction and generation of phagemids
displaying ecotropic retroviral envelope
-
The SurfZAPÔ Vector system (Stratagene) was used for the
construction of ecotropic retroviral envelope displaying
phagemids. The system is based on a bacteriophage lambda
vector in which the inserted sequences are displayed as N-terminal
fusion proteins with the minor coat protein pIII
(gpIII). Five different ecotropic envelope fragments varying
in length (See figure 1) were amplified by PCR from the
construct pMLV-K which contains a complete ecotropic
retroviral genome (Moloney) cloned into pBR322 (Miller and
Verma, 1984). The primers that were used for the construction
of envelope display phagemids are summarised in table 1. The
resulting constructs are designated gpIII/env1, gpIII/env2,
gpIII/env3, gpIII/env4 and gpIII/env5. The PCR reaction was
performed with 20 picomoles sense primer, 20 picomoles
antisense primer, 1 nanomoles dNTP mixture, 1 nanogram pMLV-K,
1x buffer B (2 mM MgSO4, 5x buffer provided with the
enzyme) and 1 unit of ELONGASEÔ Enzyme mix (Life
Technologies), adjusted to 50 ml with H2O and 25 ml mineral
oil on top. The following program was used on a Biometra
TRIO-Thermoblock: 30 sec 94°C, 30 cycles of 30 sec 94°C, 30
sec 55°C (construct 1 and 4) or 60°C (construct 5) or 68°C
(construct 2 and 3) and 30 sec 68°C, ending the cycles with a
single step of 10 min 72°C. The PCR fragments were purified
using Geneclean DNA purification kit followed by digestion
with NotI and SpeI and cloned into the SurfZAP vector arms
(NotI (left arm), SpeI (right arm)). This was followed by in
vitro packaging of the constructs using Stratagene's
Gigapack® Gold packaging extracts and amplification of the
resulting lambda phage in XL1-blue E. coli bacteria. The
lambda phages were converted to pSurfscript SK(-) phagemids
by co-infection of XL1-blue bacteria with lambda phages and
ExAssist helper phage. After amplification of the excised
phagemids displaying phages were prepared as described in
example 2. For further details the reader is referred to the
instruction manual of the SurfZAP vector system (Stratagene,
cat# 240211 and 240611). Expression of the gpIII/ envelope
fusion proteins was tested by Western blot analysis with
lysates made from bacteria and phage. To prepare the
bacterial lysates Luria Broth medium containing 2% glucose
and 100 mg/ml ampicillin was inoculated from a glycerol stock
containing the gpIII/env constructs in SOLR bacteria. After
14-16 hours of shaking (225 rpm) at 37°C 1 ml of the culture
was spun for 30 sec at 14000 rpm at 4°C (Eppendorf centrifuge
#5402). The bacterial pellet was washed in 0.5 ml icecold 1M
Tris-HCl pH 7.5 followed by resuspension in 200 ml H2O and
100 ml 3x Laemmli-buffer. After 20 sec of vortexing the
suspension was incubated for 5 min at 95°C and subsequently
sonicated for 30 sec with an amplitude of 10 microns
(Sonyprep 150). After centrifugation for 10 min at 14000 rpm,
4°C the lysates were transferred to fresh tubes and tested by
Western blot analysis (20 ml per sample). The phage lysates
were prepared by mixing 100 ml phages (prepared as described
in the next paragraph) with 50 ml 3x Laemmli-buffer followed
by incubation for 5 min at 95°C. For the Western blot
analysis 25 ml per sample was used.
The five ecotropic retroviral envelope fragments for phagemid
construction were obtained from the construct pMLV-K by PCR
(see figure 2 panel a), digested with NotI and SpeI and
ligated into the SurfZAP vector. The gpIII/envelope
constructs were tested by restriction analysis and sequencing
of the DNA inserts (BaseClear, Leiden) and found to be
correct. All constructs had the original signal peptide of
the viral sequence replaced by an E.coli/m13 signal peptide,
the pelB leader sequence. Furthermore carboxy terminal
linking of the envelope sequence to the pIII protein was done
through a GGGGS linker sequence (see figure 1, table 1 and
2). The various constructs differ in the sequences amino or
carboxy terminal of the variable regions A and B, the regions
involved in receptor recognition. Construct 1 contains the
full gp70 sequence until the protease cleavage site,
including the variable regions A and B and the polyproline
hinge region (amino acid residues 34-468). Construct 2 is
like construct 1 but stops just after the polyproline region
(amino acid residues 34-308). Construct 3 is like constructs
1 or 2 but stops before the beginning of the polyproline
region (amino acid residues 34-266). Construct 4 encompasses
the variable region A and B (amino acid residues 83-213).
and construct 5 only region A the key receptor binding domain
(amino acid residues 83-163). Numbering of amino acid
sequences was done according to the unprocessed envelope
sequence as deposited in the Swiss prot database with
accession number P03385 and starting from the viral signal
peptide. For further details see table 2.
-
Display of the fusion proteins was analysed using bacterial
and phage lysates by SDS-PAGE and Western blotting using a
monoclonal antibody against the gene III protein of m13. The
results of such a Western blotting experiment are shown in
figure 2. The bacterial lysates show bands corresponding to
the expected length of the open reading frames of the DNA
constructs (figure 2 panel a and figure 1) suggesting that
the fusion proteins are at least expressed correctly and are
stable in the E.coli host. Display of the envelope fusion
proteins was studied by performing a Western analysis similar
as for the corresponding bacterial lysates but with
approximately 6 x 109 cfus per lane of lysed phage
preparations. The results of this analysis indeed show
expression of envelope fusion proteins. Furthermore the
expression patterns for the five constructs reveal in size
descending protein bands in line with the descending pattern
of the DNA constructs.
Example 2 binding of displayed ecotropic envelope to it's
natural receptor mCAT1
-
To study mCAT1 receptor binding of the envelope displaying
phages both 911-pcDNA3 and 911-mCAT1 cell lines were used.
For making these two cell lines the human E1 immortalised
cell line 911 (Fallaux et al., 1996) was transfected either
with the mCAT1 expression construct pcDNA3-mATRC1 (Albritton
et al., 1993; Albritton et al., 1989) or with the pcDNA3
vector itself. The transfection was followed by selection for
neomycine resistance in 1 mg/ml Geneticin (Life technologies)
and by cloning the 911-mCAT1 cells. A cloned cell line
designated 911-mCAT1-3H7 was isolated which showed high
expression of mCAT1 mRNA as determined by Northern blot
analysis. Functionality of the ecotropic retroviral receptor
mCAT1 in 911 cells was shown by infection of these cells with
a ecotropic retroviral vector and subsequent measurement of
the marker gene. Control 911-pcDNA3 cells could not be
transduced and 911-mCAT1 cells could be transduced as
effectively as murine 3T3 cells. Both cell lines were
maintained in Dulbecco's modified eagle medium (DMEM) (Life
technologies) supplemented with 10% foetal bovine serum
(FBS).
-
To prepare displaying phages Luria Broth medium containing 2
% glucose and 100 mg/ml ampicillin was inoculated from a
glycerolstock containing the gpIII/env constructs in SOLR
bacteria. After 14-16 hours shaking (225 rpm) at 37 °C the
culture was diluted to OD600=0.05 and incubated for 1.5-2
hours at 37 °C with shaking until OD600=0.5. Infection with
helper phage was performed by adding 1 x1010 pfu VCSM13
(Stratagene) per ml culture and incubating at 37 °C for 30
min. Afterwards the cells were spun down for 10 min at 4000
rpm (Heraeus, Minifuge RF), resuspended in LB, 50 mg/ml
kanamycin and 100 mg/ml ampicillin and incubated for 14-16
hours at 37 °C with shaking. Phage precipitation was carried
out by spinning the cells for 20 min at 4000 rpm, adding 1/5
volume 20 % Polyethylene Glycol 8000 (PEG), 2.5 M NaCl to the
supernatant and incubating 1 hour on ice. The phages were
spun down for 20 min at 4000 rpm and resuspended in PBS.
Residual bacteria were spun down for 2 min at 14000 rpm at 4
°C (Eppendorf centrifuge #5402). The supernatant was
precipitated by PEG for the second time by adding 1/5 volume
20 % PEG, 2.5 M NaCl and incubating on ice for 20 min
followed by centrifugation for 20 min at 4000 rpm at 4 °C and
the pellets were resuspended in PBS. Residual bacteria were
spun down for 2 min at 14000 rpm at 4 °C. Phage supernatants
were stored at 4 °C until further use. The titer was
determined by adding an equal amount of XL1-blue bacteria in
10 mM MgSO4, OD600=0.5 to several dilutions of the phage
preparations and plating this mix on LB plates containing 2 %
glucose and 100 mg/ml ampicillin. After 14-16 hours
incubation at 37 °C the titers were determined using the
following formula: number of colonies x dilution factor x
1000/ volume plated = cfu/ml.
-
In a 15 ml polypropylene tube 0.5-1 x106 cells were
preincubated in 3 ml PBS containing 10% FBS and 2% non-fat
milkpowder (Brand name Elk) for 30 min at room temperature on
a slow turning rotator. At the same time 1 x1010 - 1x1011
envelope displaying phages were preincubated in 3 ml of PBS,
10% FBS and 2% non-fat milkpowder (Brand name: Elk) for 30
min at room temperature on a slow turning rotator.
Subsequently, the cells were spun down for 5 min at 1500 rpm
(Heraeus, Minifuge RF) and resuspended in the preincubated
phage solution. After 1 hour of rotation at room temperature,
the cells with the bound phage were washed 10 times with PBS,
10% FBS and 2% non-fat milkpowder (Elk) and twice with PBS.
Then the cell pellet was resuspended in H2O and an equal
volume of 200 mM Triethylamine (Sigma) was added dropwise
while vortexing. After 5 min of incubation at room
temperature, the suspension was neutralised by dropwise
addition of Tris-HCl pH 7.5 (final concentration 0.33 M)
while vortexing. The cells were spun down for 5 min at 14000
rpm (Eppendorf centrifuge) and supernatant containing the
phage was transferred to a new tube. The phages were stored
at 4°C and titered as described in example 1 and 2.
-
Following the protocol for preparation of gpIII/envelope
displaying the titer of the phage batches varied from 2.7
x1010 to 1 x1012 colony forming units (cfu) per ml (see table
3). Therefore display of the gpIII/ envelope fusion proteins
does not strongly interfer with the production of phage
particles. We now wanted to know whether the displayed
envelope moieties in fact are functional and behave as
retroviral gp70 and thus bind to the mCAT1 receptor for
ecotropic retrovirus. For this purpose we carried out binding
experiments using human cells that express the mCAT1 receptor
protein. For these binding experiments 1 x1010 -1 x1011 phages
displaying ecotropic retroviral envelope protein or parts
thereof were incubated with 0.5-1 x10 6 911 cells stably
expressing the mCAT1 receptor or 911 cells stably transfected
with the pcDNA3 vector. The number of envelope fusion phages
eluted from control cell line 911-pcDNA3 averaged around 1
x104 cfus. On 911-mCAT1 cells different elution behaviour was
visible, from 2.3 x104 to 3.6 x106 cfus. For gpIII/env 4 and
gpIII/env 5 the number of eluted phages after selection on
911-mCAT1 cells was comparable to the number of eluted phages
after selection on 911-pcDNA3 cells. Phages of the gpIII/env
1, gpIII/env 2 and gpIII/env 3 constructs show respectivily
11, 97 and 26 times more eluted phage from 911-mCAT1 cell
line as compared to the 911-pcDNA3 cell line (figure 3).
Furthermore the stability of envelope displaying phages
(gpIII/env3) was addressed by measuring binding to mCAT1 and
titer on bacteria at various periods of time after
preparation. After 1 day, 2 weeks and 4 weeks after
preparation no significant differences were observed. These
results show that 3 of the 5 constructs clearly display
functional ecotropic envelope. These phages can thus be used
to make libraries of mutants that can be used to develop
preferred envelopes for applications in gene therapy,
molecular biology and virology.
Example 3. Inhibition of envelope phage binding by ecotropic
retroviral particles
-
Functional phage display of ecotropic envelope also implies
that mCAT1 expressing cells infected or preincubated with
ecotropic retrovirus should decrease the binding of the
envelope displaying phages to mCAT1. To confirm the latter
both control and mCAT1 expressing cells were preincubated
with replication competent ecotropic retrovirus produced on
murine fibroblasts (3T3-RCR).This was done as follows:
Two days before the 911-pcDNA3 and 911-mCAT1 cells were used
for the binding experiment they were seeded in a T25 cm2
flask with DMEM containing 10% FBS and 4 mg/ml polybrene
(Hexadimethrine bromide, Sigma). After 12-16 hours incubation
at 37 °C and 10 % CO2 the medium was discarded and replaced
with 4 ml replication competent ecotropic retrovirus produced
on murine fibroblasts (NIH 3T3) or 4 ml DMEM containing 10%
FBS (control). After infection of 12-16 hours at 37 °C and 10
% CO2 the cells were harvested by trypsinisation and used in
envelope phage binding experiments.
-
Using this protocol the number of eluted gpIII/env 3 phages
from mCAT1 expressing 911 cells was decreased on average 35 %
(figure 4). This further confirms that the phages displaying
ecotropic envelope indeed have features similar to the gp70
moiety displayed on a native ecotropic retroviral virion.
Example 4 Generation of an envelope display library
encompassing mutant envelope sequences
-
The plasmid DNA constructs gpIII/env2 and gpIII/env3 in
pSURFscript were used to construct two libraries of
randomised, mutant envelope sequences. Two different
techniques to generate pools of mutants can be followed:
- (1) The gpIII/env2 plasmid DNA was passed through the
mutagenic E.coli strain XL1-Red (genotype: endA1 gyrA96 thi-1
hsdR17 supE44 relA1 lac mutD5 mutS mutT Tn10 (Tetr)). The
XL1-Red strain is deficient in three of the primary DNA
repair pathways in E.coli, the mutS, mutD and mutT, making
its mutation rate approximately 5,000-fold higher than that
of its wild-type parent (Greener et al., 1996; Greener et
al., 1997). GpIII/env2 or gpIII/env3 plasmid DNA was used to
transform competent XL1-Red cells that were purchased from
Stratagene (catalogue no: 200129) with a transformation
efficiency of approximately 106 cfus/mg pUC18 DNA.
Transformed cells were plated on large LB-agar petri-dishes
with ampicillin (100 mg/ml) and incubated at 37 °C overnight.
Ampicillin resistant colonies were scraped off the plates and
used to inoculate an 1000 ml Erlenmeyer flask with 250 ml LB
and ampicillin (100 mg/ml). The Erlenmeyer flasks were
transferred to a shaking incubator set at 37 °C and incubated
overnight or as long as it takes to get sufficient bacteria.
The following day the cultures were subjected to a plasmid
DNA isolation procedure using the Qiagen plasmid DNA column
method.
The isolated plasmid DNA is verified using Notl, SpeI and
PinA1 enzyme digestions and DNA agarose gel electrophoresis. -
-
As an example two strategies can used to generate envelope
displaying libraries based on XL1-Red mutagenized gpIII/env2
and 3 DNA: (A) The XL1-Red propagated and mutagenized DNA as
such was used directly to generate libraries of envelope
displaying phages as described under examples 1 and 2 using
XL1-Blue competent bacteria.
(B) The XL1-Red mutagenized plasmid DNA of gpIII/env2 and 3
are digested with Not1 and SpeI followed by isolation of the
Notl/SpeI 869 basepairs (gpIII/env2) and 743 basepairs
(gpIII/env3) fragments using DNA agarose gel electrophoresis
and Qiaquick gel extraction method (Qiagen). These fragments
are ligated back into gpIII/env or 3. This was done by
digesting gpIII/env2 or 3 propagated in DH5alfa E.coli with
NotI and SpeI and isolation of a 3631 basepair fragment. The
pools of ligated molecules can then be used to generate
envelope display libraries by transformation into competent
XL1-Blue cells and subsequent steps as described in example
1.
- (2) The second technique that was used to generate randomised
envelope displaying phage libraries was shuffle PCR (Stemmer,
1994a; Stemmer, 1994b). Shuffle PCR generates libraries of
mutant genes by recursive sequence recombination. This is
done by digesting a PCR amplified target sequence with DNA
polymerase I followed by isolation of smaller DNAs I digested
fragments (30-700 basepairs). The latter is followed by Taq
polymerase mediated reassembly of the full length fragment
without the addition of any single strand synthetic
oligonucleotides. Reassembled, shuffled target sequence is
then amplified and cloned back into the appropriate vector.
For the generation of a library of phages displaying
randomised envelopes sequences using shuffle PCR, the
following PCR was performed using pMLVK (Miller and Verma,
1984) plasmid DNA made in DH5alfa E,coli or XL1-Red E.coli
bacteria as a template per 100 ml total reaction volume. For
construction of a shuffled gpIII/env2 library the primer-pair
ecoenv17/ecoenv12 was used and for gpIII/env3 shuffled
libraries the primer-pair ecoenv17/ecoenv15 (see table 1).
The PCR reactions were performed with 40 picomoles sense
primer, 40 picomoles antisense primer, 2 nanomoles dNTP
mixture, 1 nanogram pMLV-K or gpIII/env2 or 3, lx SuperTaq
buffer and 0.5 units of SuperTaq thermostabile polymerase
(both from HT Biotechnology, UK) adjusted to 100 ml with H2O
and 25 ml mineral oil on top. The following program was used
on a Biometra TRIO-Thermoblock: 30 sec 94°C, 30 cycli of 30
sec 94°C, 30 sec 68°C and 30 sec 72°C, ending the cycles with
a single step of 10 min at 72°C. Amplified DNA was purified
using Qiaquick PCR purification cartridges and checked for
appropriate size (869 basepairs for ecoenv17/ecoenv12 and 743
basepairs ecoenv17/ecoenv15) by agarose gel electrophoresis.
Five micrograms of the amplified DNA was then subjected to a
digestion with 0.15 units of DNAseI (Sigma D-7291)(DNAseI
concentration of 0.0015 Units/ ml) in 50 mM Tris-HCl pH 7.4,
1 mM MgCl2. Digest for 5 minutes at 25 °C, put on ice and
verify extent of digestion by loading an aliquot on a 2 %
agarose gel in 1 x TAE together with a lane with a suitable
DNA marker to determine size range of generated fragments. If
sufficient material is generated in the desired size range of
30-700 basepairs the tube is incubated for 10 minutes at 65
°C to inactivate the DNAse I enzyme. Otherwise incubate an
additional 5 minutes at 25 °C and repeat the above. Once
sufficient DNAse I digested material has been generated all
DNAse I treated material of the two fragments can be loaded
on a gel. and the fragments of 50-300 basepairs isolated from
the gel using a Qiaquick gel extraction kit or equivalent.
One hundred to 2000 nanograms in 100 ml final volume of
fragment is then subjected to a reassembly step with a hot
start of 1 minute at 94 °C and 45 cycles of 30 seconds at 94
°C, 30 seconds at 50 or 55 °C and 30 seconds at 72 °C. The
composition of the buffer is identical to that used for
amplification of the primary PCR products using
ecoenv17/ecoenv12 and ecoenv17/ecoenv15 primer pairs except
for the absence of any ecoenv primer. To monitor the extent
of reassembly samples can be taken at 25,30,35, 40 and
finally 45 cycles. Extent of reassembly is visualised by
loading on a 1-2% agarose gel and comparison to the size of
the primary PCR product, input DNAseI fragments. Reassembly
is followed by amplification of reassembled, shuffled
fragment to sufficient amounts of DNA for cloning back into
the envelope phage display vector. For this purpose various
amounts of reassembled DNA are subjected to the same PCR
reactions used for generation of the primary PCR reactions
and thus using the same pair of primers. Alternatively for
higher yields of shuffled fragment Elongase (Life
technologies) is used (see for experimental details example
1). The reassembled shuffled pool of fragments (approximately
869 basepairs in length for ecoenv17/ecoenv12 (gpIII/env2)
and approximately 743 basepairs for ecoenv17/ecoenv15
(gpIII/env3)) are purified using Qiaquick PCR purification
cartridges and digested with NotI and SpeI or with NotI and
PinA1 followed by isolation of the digested fragments using
agarose gel electrophoresis. This results in 4 different
pools of fragments that can be ligated into NotI/SpeI or
NotI/PinAI digested gpIII/env2 or gpIII/env3 vector fragment.
The ligation mixtures are then used to generate phage
libraries displaying shuffled envelope molecules as described
under examples 1 and 2. Digestion with NotI and SpeI or NotI
and PinA1 results in libraries displaying varying parts of
the gp70 sequence as a shuffled library of mutants. The
skilled artisan will be able to apply the teaching of the
above to the generation of mutant envelope molecules by
incubating target cells of any origin including but not
limited to human CD34+ cells enriched for hemopoietic stem
cells with the various libraries and repeating these rounds
of selection until envelope displaying phages have been
selected for that are optimised for binding and entry into
the desired target cells. These mutant envelope molecules or
parts thereof can then be ligated into full length mammalian
ecotropic envelope expression constructs and introduced in
cell lines expressing and containing all the sequences
necessary for the generation of infectious and functional
retroviral particles including but not limited to cell lines
that express murine leukemia gag-pol constructs and a
retroviral vector containing long terminal repeats (LTRs),and
retroviral RNA packaging signals such as those vectors
described in WO96/35798. The mutant envelopes made according
to the subject material of this invention can also be used to
pseudotype vectors other than murine leukemia retroviral
vectors including but not limited to lentiviral vectors
(Naldini et al., 1996a).
-
Example 5
-
FAb phage display to select CAT1 binding antibody molecules
for for example incorporation in a mutant protein.
-
To isolate antibodies that bind to CAT we also employed
phages displaying FAb fragments encompassing the light and
heavy variable and constant regions. A FAb phage display
library was constructed in phage display vector pCES1 a
vector derived from pCANTAB6 (McGuiness et al., 1996). The
library was constructed in the filamentous E. coli phage m13
and the FAb sequences are displayed partly as N-terminal
fusion proteins with the minor coat protein pIII. Two targets
were used to select for peptide displaying phages which bind
to the third extracellular domain of CAT. First the predicted
third extracellular domain of CAT was synthesized as a
synthetic peptide by Neosystem, Strassbourg, France. The N-terminus
of this peptide was biotinylated and followed by
three amino acid linker residues KRR, followed by the
predicted sequence of the third extracellular domain.
Secondly we generated cell lines that overexpress CAT as
judged by steady state mRNA expression levels. A CAT
expression construct which is a pcDNA3 based expression
construct of CAT cDNA was employed to transfect CAT
expressing cell lines followed by selection for neomycine
resistance in 1 mg/ml of G418 (Geneticin, Life Technologies,
Inc).
-
To select for FAb displaying phages that bind to the
putative third extracellular domain of CAT as expressed on
human cells the following selection procedure was employed.
Four rounds of selection on biotinylated CAT peptide followed
by three rounds of selection on CAT overexpressing cells. For
selection on biotinylated CAT peptide 250 ml of FAb library
(or eluted phage from the previous round) was mixed with 250
ml 4% Marvel in PBS and equilibrated while rotating at RT for
1 hour. Subsequently biotinylated CAT peptide (20-500 nM in
H2O) was added. This mix was incubated on the rotator at RT
for 1 hour before 250 ml equilibrated streptavidin-dynabeads
in 2% Marvel in PBS was added. After incubation on a rotator
at RT for 15 min the beads with the bound phage were washed 5
times with PBS/2% Marvel/0.1% Tween, 5 times with PBS /0.1%
Tween and 5 times with PBS. Then the phage were eluted by
incubation with 0.1M Tri-ethyl-amine on a rotator at RT for
10 min and neutralised in 1 M Tris-HCl pH 7.4. The eluted
phage were titered and amplified in TG1 before the next
selection. For selection on CAT expressing cells, the cells
were harvested at a confluency of about 80 % and suspended in
PBS/10 % FBS/2 % Marvel to a final concentration of at least
3x106 cells/ml. This cell suspension was incubated for 30 min
on a rowing boat mixer (or rotator), while at the same time
phage were also preincubated in PBS/10 % FBS/2 % Marvel. Then
the cells were centrifuged, resuspended in the preincubated
phage solution and incubated on a rowing boat mixer (or
rotator) for 1 hour. Afterwards the cells were washed 10
times with PBS/10 % FBS/2 % Marvel and twice with PBS. The
cells were centrifuged and resuspended in 0.6 ml water.
Subsequently 0.6 ml 200 mM triethylamine was added (dropwise
while vortexing). After 5 minutes the suspension was
neutralised by adding 0.6 ml of 1 M Tris-HCl pH 7.4 (dropwise
while vortexing).After cenrifugation (5 min, 14000 rpm) the
supernatant was transferred to a new tube and titered and
amplified in TG1 before the next selection. The results of
these experiments show that the ratio of input over output
increases upon selection on CAT peptide indicative of
selection for CAT peptide binding phages. When selection on
CAT positive cells was started the ratio dropped and slightly
increased in the last round on CAT expressing cells.
-
The pools of the last 3 rounds were tested for binding
to the biotinylated CAT peptide in a CAT specific ELISA and
also for cell binding by flow cytometric analysis. After the
last round of selection on cells the pool of FAb phages still
binds to the biotinylated CAT peptide. Flow cytometric
analysis showed that this pool also binds to CAT
overexpressing cells. From this pool clones were analysed by
fingerprint analysis. Double strand phagemid DNA was prepared
and used to determine the nucleotide and deduced amino acid
sequence of the displayed variable heavy and light chains by
clones binding to CAT. The relevant sequences or parts
thereof of this immunoglobulin can be incorporated in viral
or non-viral mutant proteins that mediate binding and entry
to cells and thus create gene transfer vehicles that enter
cells through CAT.
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Albritton, L.M., Kim, J.W., Tseng, L. and Cunningham, J.M.
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Annex to the description
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